Predicting prognosis and treatment response of breast cancer patients using expression and cellular localization of N-myristoyltransferase

ABSTRACT

High levels of nuclear NMT1 are associated with longer relapse free survival in ERα positive breast cancer patients. Both low levels of cytosolic and nuclear NMT1 correlated to very poor clinical outcomes. NMT2 also plays an important function in breast cancer signalling, regulated through phosphorylation. For example, NMT2 phosphorylation status is a key element in the progression of ER+ breast cancer cells. Specifically, nuclear localization of NMT2 is associated with poor outcomes in breast cancer patients.

PRIOR APPLICATION INFORMATION

The instant application is a 371 of PCT CA 2022/050808, filed May 20,2022, which claimed the benefit of U.S. Provisional Patent Application63/190,905, filed May 20, 2021, entitled “PREDICTING PROGNOSIS ANDTREATMENT RESPONSE OF BREAST CANCER PATIENTS USING EXPRESSION ANDCELLULAR LOCALIZATION OF N-MYRISTOYLTRANSFERASE”, the entire contents ofwhich are incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

The human N-myristoyltransferase (NMT) exists in two forms—NMT1 andNMT2. The gene for human NMT1 is located on the long arm of chromosome17 and the alterative isoforms appear to be splicing variants(Selvakumar P et al., Prog Lipid Res 2007, 46(1):1-36) whereas NMT2 islocated on chromosome 10. Previous studies have indicated NMT to besolely cytosolic; however, recently, it has been demonstrated that10-50% may be associated with a particulate fraction (Boutin J A, CellSignal 1997, 9(1):15-35).

The majority of breast cancers arise from epithelial cells lining theducts of the breast tissue, and are thus categorized as carcinomas(Sharma, G. N., et al., Journal of Advanced Pharmaceutical Technology &Research, 2010. 1(2): p. 109-126). Central to the cell signalling inmost breast carcinomas is estrogen signalling. Estrogen signalling isregulated through the interplay between the two distinct estrogenreceptor isoforms (ER α and ER β) and their respective splice variants(Heldring, N., et al., Physiol Rev, 2007. 87(3): p. 905-31). Both ERsare members of the nuclear receptor family of transcription factors,dimerizing upon ligand binding and subsequently localizing to thenucleus to initiate gene transcription (Tamrazi, A., et al., MolEndocrinol, 2002. 16(12): p. 2706-19). ER activated genes are regulatedby regions of DNA collectively known as estrogen response elements(EREs) (Klinge, C. M., Nucleic Acids Research, 2001. 29(14): p.2905-2919). The primary ER ligand, the steroid hormone estrogen, is apotent morphogen responsible for driving the proliferation of epithelialbreast tissues following its binding to EREs, as well as a range ofother effects in men and women including those on the cardiovascular,musculoskeletal, immune, and central nervous systems (Gustafsson, J. A.,Trends Pharmacol Sci, 2003. 24(9): p. 479-85). Specifically, ER mediatedtranslation produces proteins essential in key processes in breastcancer development, including cell division, survival, and angiogenesis(Osborne, C. K., et al., Clin Cancer Res, 2001. 7(12 Suppl): p.4338s-4342s). ERs may also participate in “nongenomic” signallingthrough interaction with proteins in other growth signalling pathways(Losel, R. M., et al., Physiol Rev, 2003. 83(3): p. 965-1016).

Of the different biological estrogen forms, 17β-estradiol (E2) is themost potent version and is the form most frequently involved in breasttissue tumorigenesis (Simpson, E. R., J Steroid Biochem Mol Biol, 2003.86(3-5): p. 225-30). ER α and ER β are known to have some distinct anddivergent functions following the E2 response. This is especiallyevident at the promoters of important proliferation genes, in which ER αand ER β often have opposing effects (Liu, M. M., et al., J Biol Chem,2002. 277(27): p. 24353-60). Of the two ER isoforms, ER α overexpressionis associated with breast cancer: over half of primary breast cancersexhibit ER α overexpression and approximately 70% of these are sensitiveto anti-estrogen therapy (Ali, S. and R. C. Coombes, J Mammary GlandBiol Neoplasia, 2000. 5(3): p. 271-81). Typically, these ER positivebreast cancers are treated with either selective estrogen receptormodulators (SERMs), such as tamoxifen, or they are treated witharomatase inhibitors, including anastrozole, exemestane and letrozole(AIs). SERMs generally bind to the ER and act as a competitive inhibitorto block estrogen growth signalling; however, tamoxifen (and othertriphenylethylene drugs) does behave as a partial agonist, displayingtissue-selective pharmacology. In fact, evidence suggests that tamoxifenactivates the ER, with the subsequent conformational changes oftamoxifen-bound ER resulting in the preferential recruitment ofcorepressor complexes that lead to gene silencing. Currently, tamoxifenremains the gold standard treatment for primary breast tumors. Due tosome of the anti-proliferation effects of ER β signalling, ER β agonistshave also been considered in the treatment of some breast cancers(Montanaro, D., et al., J Mol Endocrinol, 2005. 35(2): p. 245-56). Theaforementioned treatments are examples of endocrine therapy (also knownas hormonal therapy).

ER+ Positive Breast Cancer

Hormone receptor positive breast cancer cells overexpress ER and/or PR,are dependent on the production of endogenous estrogen or progesteroneto activate hormone dependent signalling pathways which regulatecellular proliferation rates. The ER has both nuclear (genomic) andnon-nuclear (non-genomic) functions and is the major driver of themajority of breast cancers. ER+ breast cancers account for approximately75% of all diagnosed breast cancer cases (C. K. Osborne and R. Schiff,Annu Rev Med, vol. 62, pp. 233-247, 2011). As discussed above, ER existin two isoforms, ERα and ERβ, which belong to the steroid hormonereceptor family of nuclear receptors. ERα is the receptor found to beoverexpressed in ER+ breast cancer cells and therefore serves as aprimary biomarker for ER+ breast cancer prognosis (M. H. Zhang et al.,Biomed Rep, vol. 2, no. 1, pp. 41-52, January 2014).

Selective Endocrine Receptor Modulators (SERMs)

As discussed above, there are primarily three classes of agents used totreat ER+ breast tumors: Selective endocrine receptor modulators (SERMs,such as tamoxifen), estrogen synthesis inhibitors (aromatase inhibitors(AIs), such as anastrozole) and selective endocrine receptor downregulators (SERDs, such as fulvestrant) (M. Giuliano et al., The Breast,vol. 20, pp. S42-S49, October 2011). Breast cancer tumors are typicallyremoved by surgery and/or treated with chemotherapy, radiation, andvarious adjuvant drug therapies such as SERMs. Tamoxifen, which acts asan ER antagonist, competitively inhibiting endogenous estrogen moleculesfrom binding to the ER active site, is considered to be one of the mosteffective forms of hormonal therapy and is the most commonly prescribedSERM for ER+ breast cancer patients.

Despite the relative success of endocrine therapies in treating breastcancer, de novo and developed resistance to these therapies (endocrineresistance) are still issues of major concern. Almost 50% of breastcancer patients with primary tumors exhibit de novo resistance to firstline tamoxifen treatments, with tamoxifen sensitive individuals oftenacquiring resistance to the drug after an initial positive response.Actual ER expression loss accounts for only a small fraction (10%) ofendocrine resistance cases in primary and metastatic tumors (Sighoko,D., et al., Oncologist, 2014. 19(6): p. 592-601). Aberrant PI3K/AKT/mTORsignalling occurs in about 70% of breast cancers, with signallingmolecules downstream of the IGF1R receptor also contributing toendocrine resistance, including mutations to the PIK3CA, AKT1, AKT2,PDK1, PTEN and INPP4B genes (Fu, X. et al., Breast, 2013. 22 Suppl 2: p.S12-8).

The PI3K/AKT/mTOR signalling pathway is involved in regulating glucosemetabolism, angiogenesis, cell survival, proliferation, and migration,and is often dysregulated in many types of cancer, including breastcancer. The PI3K pathway is triggered by insulin, and growth factorssuch as EGF, FGF and IGF-1. AKT, which is central to the PI3K pathway,is a serine/threonine protein kinase and proto-oncoprotein (Bellacosa,A., et al., Adv Cancer Res, 2005. 94: p. 29-86). AKT is normallycytoplasmic, but locates to the inner cell membrane by AKT's PlekstrinHomology (PH) domain binding to PIP3, exposing activation sites on AKTfor hydroxyl group phosphorylation. Primary phosphorylation of AKT sitesfor activation are at T308, phosphorylated by phospho-inositidedependent kinase 1 (PDK1), and S473, which is phosphorylated by mTORcomplex2 (mTORC2). In ER+ breast cancer cell lines, upon phosphorylationat both T308 and S473, AKT is fully activated and translocates to thecytoplasm, nucleus or other sub-cellular compartments, where itphosphorylates other substrates. A downstream target of AKT is mTOR,which is a kinase that regulates the cellular processes of cell growth,proliferation in response to nutrient/energy availability, signallingstimuli and translation of protein. it has been demonstrated that AKToverexpression leads to decreased NMT activity (Shrivastav, A., et al.,J Pathol, 2009. 218(3): p. 391-8). Preliminary studies in our lab havedemonstrated that mTOR interacts with and potentially phosphorylatesNMT1.

NMT Subcellular Localization

Despite the overlapping targets of NMT1 and NMT2 and their variants,they appear to have different roles in cell apoptosis, during which themyristoylated proteome undergoes drastic changes (Perinpanayagam, M. A.,et al., Faseb j, 2013. 27(2): p. 811-2). Ablation of NMT2 has been shownto induce a 2.5× greater rate of apoptosis over NMT1 knockdown inSK-OV-3 ovarian carcinoma cells (Ducker, C. E., et al. Mol Cancer Res,2005. 3(8): p. 463-76).

Depletion of NMT2 also yielded a shift in BCL family proteins towards astate of apoptosis. These findings support the notion that NMT1 may bethe primary NMT involved in driving apoptosis, with NMT2 associatingwith suites of pro-growth signalling proteins. The same study found thatdual depletion of NMT1 and 2 was lethal and that this effect was p53independent. The line of division between these enzymes' roles may bedrawn through their dynamic and differential localization duringapoptosis, among other cell states. Perinpanayagam et al demonstratedthat both NMT isoforms are cleaved by caspases during apoptosis, inwhich NMT1 and NMT2 localization changes significantly ((Perinpanayagam,M. A., et al. Faseb j, 2013. 27(2): p. 811-2). NMT1 was shown to becleaved at Aspartic Acid-72 by either effector caspase 3 or extrinsiccaspase 8; NMT2 was shown to be cleaved at Aspartic Acid-25 by effectorcaspase 3. Caspase-3 is an executioner caspase, which catalyzes thecleavage of many cellular proteins involved in programmed cell death(Nicholson, D. W., Cell Death Differ, 1999. 6(11): p. 1028-42).Following caspase cleavage, which leaves behind a poly-basic domainstretch, a greater population of NMT1 translocated to cytoplasm (55%)from membrane bound whereas NMT2 underwent an even greater shift inlocalization following caspase cleavage which removed a negativelycharged domain, rendering 80% of NMT2 membrane bound as opposed to 62%cytoplasmic prior to caspase cleavage.

Interestingly, serine residues (which are capable of beingphosphorylated) within the human NMT isoforms appear to be homologousbetween different species and between the isoforms themselves.Specifically, serine 47 of NMT1 is similar in relative position toserine 38 of NMT2, with respect to the poly-lysine region in theN-terminus. Additionally, serine 68 which follows the poly-lysine domainof NMT2 is similar in position with serine 73 of NMT1, which has alsobeen identified as phosphorylated in NMT1 following an ultra-deep humanphosphoproteome analysis using a human cancer cell line (Sharma, K., etal., Cell Rep, 2014. 8(5): p. 1583-94). The conservation ofphosphorylated serines on either side of the poly-lysine region of theNMTs suggests that these residues may play an important role in theregulation of NMT localization within the cell.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a methodof determining prognosis of a hormone positive breast cancer patientcomprising:

-   -   extracting a cell sample from a breast tumor of a hormone        positive breast cancer patient; and    -   determining nuclear and cytoplasmic levels of NMT1 in one or        more cells of the cell sample;    -   wherein: if cytoplasmic levels of NMT1 are high, the prognosis        is poor;    -   if nuclear levels of NMT1 are high, the prognosis is good; and    -   if both nuclear and cytoplasmic levels are low, the prognosis is        worse than poor.

According to another aspect of the invention, there is provided a methodof determining the prognosis of a breast cancer patient comprising:

-   -   extracting a cell sample from a breast tumor of a patient;    -   determining the cellular localization of NMT2 in at least one        cell of the cell sample;    -   wherein if the at least one cell of the cell sample is positive        for nuclear NMT2, the prognosis is poor, and    -   if the at least one cell of the cell sample is negative for        nuclear NMT2, the prognosis is good.

According to another aspect of the invention, there is provided a methodof determining the prognosis of a triple-negative breast cancer patientcomprising:

-   -   extracting a cell sample from a breast tumor of a patient;    -   determining the cellular localization of NMT2 in at least one        cell of the cell sample;    -   wherein if the at least one cell of the cell sample shows        positive nuclear localization of NMT2, the prognosis is poor.

According to another aspect of the invention, there is provided a methodof slowing progression or improving outcome of a breast cancer tumorcomprising:

-   -   administering an effective amount of an NMT2 serine        phosphorylation inhibitor to a patient having a cancerous breast        tumor, thereby preventing phosphorylation of NMT2 and subsequent        nuclear localization of NMT2.

According to another aspect of the invention, there is provided a methodof identifying a compound capable of inhibiting nuclear translocation ofcytoplasmic NMT2 comprising:

-   -   in an in vitro system, growing a plurality of test cells under        conditions suitable for nuclear translocation of cytoplasmic        NMT2 in the presence of a compound of interest wherein but for        the presence of the compound of interest, cytoplasmic NMT2 will        migrate into the nucleus of a respective one cell of the        plurality of cells; and    -   determining if cytoplasmic NMT2 has translocated into the        nucleus of at least one of the plurality of test cells in the        presence of the compound of interest,    -   wherein if less cytoplasmic NMT2 has translocated into the        nucleus of the at least one representative cell of the plurality        of cells than translocated into the nucleus of at least one        control cell grown under similar conditions except for the        presence of the compound of interest, the compound of interest        inhibits nuclear translocation of cytoplasmic NMT2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . High NMT1 nuclear levels correlate to longer reoccurrence freesurvival. Patients were separated into low or high categories based onthe median H-score. Kaplan Meier curves were generated to assess whetherany parameters associated with a significant change in patient survival.Reoccurrence analysis of low or high nuclear NMT1 levels.

FIG. 2 . High NMT1 nuclear levels correlate to longer reoccurrence freesurvival while both low cytoplasmic and nuclear levels of NMT1 arelinked to higher chance of recurrence. Patients were separated into lowor high categories based on the median H-score. Kaplan Meier curves weregenerated to assess whether any parameters associated with a significantchange in patient survival. Recurrence analysis of low and high nuclearand cytoplasmic NMT1 levels.

FIG. 3 . Low NMT1 nuclear levels correlate with higher rate of death dueto breast cancer or recurrence. Patients were separated into Low or Highcategories based on the median H-score. Kaplan Meier curves weregenerated to assess whether any parameters associated with a significantchange in patient survival. Death due to breast cancer or recurrenceanalysis of low and high levels of nuclear NMT1.

FIG. 4 . Patients with high NMT1 nuclear levels were less likely to diedue to breast cancer or recurrence. Patients were separated into low orhigh categories based on the median H-score. Kaplan Meier curves weregenerated to assess whether any parameters associated with a significantchange in patient survival. Recurrence analysis of low and high nuclearand cytoplasmic NMT1 levels.

FIG. 5 . Trypan blue assay of cellular proliferation rates of MCF7 andMCF7 cells expressing phospho-site mutated NMT2 proteins. Each point isan average of three experiments and error bars represent+standarddeviation. The double asterisks (**) represents a t-test P-valuesignificance factor of <0.01 in proliferation rates between the MCF7 andMCF7-GFP-NMT2-S38E cell lines. The single asterisk (*) represents at-test P-value significance factor of <0.05 in proliferation ratesbetween the MCF7 and MCF7-GFP-NMT2-S38E cell lines. The double asterisks(**) represents a t-test P-value significance factor of <0.01 inproliferation rates between the MCF7 and MCF7-GFP-NMT2-S38A cell lines.

FIG. 6 : Crystal violet assay for cellular proliferation rates of MCF7wt and MCF7 cells expressing phospho-site mutated NMT2 proteins. Eachpoint is an average of three readings and error bars represent+standarddeviation. The single asterisk (*) represents a t-test P-valuesignificance factor of <0.05 in proliferation rates between the MCF7 andMCF7-GFP-NMT2-S38E cell lines. The double asterisks (**) represents at-test P-value significance factor of <0.01 in proliferation ratesbetween the MCF7 and MCF7-GFP-NMT2-S38E cell lines.

FIG. 7 . Expression levels of IGF1R mRNA in the four cell lines ofinterest.

FIG. 8 . Western blot PVDF membrane loaded with the four cell lines ofinterest and probed with anti-IGF1R and anti-p-actin.

FIG. 9 . Immunofluorescence images of NMT2 in (A) MCF7 cells and (B)MB-MDA 231.

FIG. 10 . TMA 16: Case with Triple Negative Breast Cancer; TMA 18-21Cases with hormone receptor positive (ER, PR positive) or triplepositive (ER, PR and Her2Neu). The patients were analyzed for theirsurvival for the first 10 years. Cases are depicted with respect to thecytoplasmic expression of NMT2.

FIG. 11 . TMA 16: Case with Triple Negative Breast Cancer; TMA 18-21Cases with hormone receptor positive (ER, PR positive) or triplepositive (ER, PR and Her2Neu). The patients were analyzed for theirdeath or recurrence for the first 10 years. Cases are depicted withrespect to the cytoplasmic expression of NMT2.

FIG. 12 . TMA 16: Case with Triple Negative Breast Cancer; TMA 18-21Cases with hormone receptor positive (ER, PR positive) or triplepositive (ER, PR and Her2Neu). The patients were analyzed for theirdeath or recurrence for the first 10 years. Cases are depicted withrespect to the nuclear expression of NMT2 (no nuclear staining: Lowzero. Positive nuclear staining: High zero).

FIG. 13 . TMA 16: Case with Triple Negative Breast Cancer; TMA 18-21Cases with hormone receptor positive (ER, PR positive) or triplepositive (ER, PR and Her2Neu). The patients were analyzed for theirdeath or recurrence for the first 10 years. Cases are depicted withrespect to the nuclear expression of NMT2 (no nuclear staining: Lowzero. Positive nuclear staining: High zero).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference.

As discussed herein, high levels of nuclear NMT1 are associated withlonger relapse free survival in ERα positive breast cancer patients,subsequently treated with tamoxifen, in univariate analysis.Interestingly, both low levels of cytosolic and nuclear NMT1 correlatedto very poor clinical outcomes, as discussed herein.

Specifically, nuclear localization of NMT1, accompanied by lowcytoplasmic expression of NMT1, was associated with relapse freerecovery following endocrine therapy. The opposite NMT1 localizationpattern, high cytoplasmic expression of NMT1, was associated withendocrine therapy resistance and bad prognosis. Thus, we hypothesizedthat NMT1 is phosphorylated downstream of the PI3K/AKT/mTOR/ERsignalling axis by mTOR, and that this phosphorylation event isimplicated in NMT1's localization to the nucleus and in good ER+ breastcancer treatment outcome following endocrine therapy. Furthermore, wepredicted that the NMT2 isozyme may also be playing an importantfunction in breast cancer signalling, and that this role may also beregulated through phosphorylation.

Furthermore, this study identified and explored potential mTOR or AKTmediated phospho-sites on the NMT1 protein, as well as identifiedputative phospho-sites on the NMT2 isozyme for future analysis.Datamining using Kinexus Phosphonet and PhosphositePlus revealed thatS40 and S47 (NMT1), and S38 and 68 (NMT2) are prime candidates forphosphorylation by mTOR.

Notably, a putative poly-lysine based nuclear localization sequence(NLS) was identified during this study within the N-termini of NMT1 andNMT2. The putative NLS in both proteins is located nearby the residuespredicated to be phosphorylated by mTOR. Phosphorylation of NMT may be apossible mechanism in which NMT containing a poly-lysine region isprimed for or inhibited from nuclear or ER localization. The presence ofS47 phosphorylated NMT1 in the nucleus suggests that thisphosphorylation event may be required for nuclear localization of NMT1;the presence of S47 phosphorylated NMT1 following DNA damage suggeststhat nuclear phopsho-NMT1 may be involved in swinging gene transcriptiontowards apoptosis.

Additionally, serine 38 and serines 66, 68 and 70 nearby the poly-lysineregion of NMT2 have been shown to be phosphorylated in cancerous tissues(Stuart, S. A., et al., A, Mol Cell Proteomics, 2015. 14(6): p.1599-615; Zanivan, S., et al., J Proteome Res, 2008. 7(12): p. 5314-26).

It is apparent that most predicted and/or observed phosphorylationevents on the NMT isozymes are concentrated around the putative NLSwithin the N-terminus, regardless of whether they are predicted to bemediated by mTOR. Phosphorylation adjacent to an NLS is a well-knownmechanism to regulate importin a mediated translocation of a protein tothe inside of the nucleus (Harreman, M. T., et al., J Biol Chem, 2004.279(20): p. 20613-21). It is plausible that the predictedphosphorylation events may stabilize the structure of the nearby NLS andkeep it exposed, as the N-termini of both NMT1 and NMT2 are normallyhighly disordered.

The S47 residue proved to be a good candidate to study the structuralstabilization of the NLS following phosphorylation. Furthermore,phospho-sites tend to be present in the disordered regions of proteins,a pattern that is apparent in NMT1 and NMT2, suggesting that thedisordered N-terminal region of NMTs is the primary regulatory region ofthe enzyme (Landry, C. R., E. D. Levy, and S. W. Michnick, Trends Genet,2009. 25(5): p. 193-7). This disordered N-terminal region is exposed andhighly flexible, making it an easy target for potentially stabilizingphosphorylation. Indeed, the multi-phosphorylation model of NMT1 inwhich the putative phospho-sites surrounding the NLS werephosphorylated, predicted that the N-terminus of the protein stabilizedto form an exposed structure resembling a Helix-Turn-Helix motif. Thistype of motif is associated with DNA binding and is common to manytranscription factors.

MCF7 breast cancer cell lines were established that express variousmutant versions of NMT1 fused to a GFP tag. These include variants witheither null mutations to the potential phospho-sites (S40A, S47A, andS256A) or phosphorylation mimicking mutations to the sites (S40E. S47E,S256E). We hypothesized that phosphorylation of these sites was involvedin shuttling NMT1 to the nucleus or endoplasmic membrane system. Thus,we expected that mutating these sites to alanine phospho-knockouts wouldresult in an NMT1-GFP fusion protein that remained primarily in thecytoplasm. Overall, our prediction was observed, with S40A, S47A andS256A expressing the fusion protein diffusely through the cytoplasm.Inversely, we predicted that at least one of the glutamic acid mutationswould result in at least one cell line that exclusively expressed NMT1in the nucleus; however, results were mixed. Localization that appearedto overlap with the nucleus was observed to a certain degree in S40,S47, and S256 phosphomimics; however, many cells in these populationsexpressed cytoplasmic NMT1. Unlike the wildtype NMT1-GFP or the alaninemutants, which expressed fusion protein evenly throughout the cytoplasm,S40E and S256E cells that expressed cytoplasmic fusion protein did so inlocalized areas. These areas often constituted a patch of expressionadjacent to the nuclear region, indicating potential localization to theER. Overall, these findings suggest that phosphorylation of all threesites may be somehow involved in translocation of NMT1 to the nucleus ornuclear membrane, with phosphorylation of S40 and/or S256 involved intranslocation of NMT1 to the ER. Within the nucleus, it is possible thatNMT1 is playing a role in transcriptional regulation.

The observation of nuclear NMT1, coupled with the identification of aputative DNA interacting NLS, sparked our interest in exploring NMT1'srole in the nucleus. We predicted that NMT1 might be interacting with amyristoylated transcriptional co-repressor, BASP1. Immunoprecipitationof BASP1 co-immunoprecipitated NMT1 protein.

Confirmation of the BASP1-NMT1 interaction within the nucleus of MCF7breast cancer cells led us to investigate a potential interaction ofNMT1 with DNA. We showed for the first time through ChIP analysis thatNMT1 appears to interact with the P21 and IGF1R growth genes, repressiontargets of BASP1. Both P21 and IGF1R expression are driving factors inthe progression of many cancers, including breast cancer. It is possiblethat the association of nuclear NMT1 with good breast cancer prognosisis due in part to repression of these and other growth genes.

Although NMT1 and NMT2 are not redundant in function, they share 77%amino acid sequence homology with analogous putatively phosphorylatedserine residues.

As discussed herein, NMT2 phosphorylation status is a key element in theprogression of ER+ breast cancer cells.

As used herein, “prognosis” refers to for example a “best estimate” ofhow a cancer will affect a patient, that is, the likely outcome of thecancer. As will be appreciated by those of skill in the art, there aremany methods for assigning a prognostic score for a particular patient,that relies on many factors. Accordingly, as used herein, “determiningprognosis” refers to the fact that the levels and/or subcellularlocation of NMT1 and/or NMT2 may represent one prognostic factor in anoverall prognosis determination. As such, referring to the prognosis asbeing “good” or “favorable” indicates that the observed levels and/orsubcellular location of NMT1 and/or NMT2 contribute positively to aprognostic score whereas referring to the prognosis as “poor” indicatesthat a negative contribution is being made to the prognostic score andreferring to the prognosis as “worse” indicates that a more negativecontribution is being made to the prognostic score. That is, a “worse”prognosis means that the prognostic score is reduced whereas prognosisas “good” or “favorable” means that the prognosis score is increased.

According to an aspect of the invention, there is provided a method ofdetermining prognosis of a hormone positive breast cancer patientcomprising:

-   -   extracting a cell sample from a breast tumor of a hormone        positive breast cancer patient; and determining nuclear and        cytoplasmic levels of NMT1 in one or more cells of the cell        sample;    -   wherein: if cytoplasmic levels of NMT1 are high, the prognosis        is poor or lowered or a prognostic score is lowered or decreased        or reduced;    -   if nuclear levels of NMT1 are high, the prognosis is good or        improved or a prognostic score is increased or improved; and    -   if both nuclear and cytoplasmic levels are low, the prognosis is        worse or the prognostic score is lowered or decreased more than        for a poor prognosis.

As discussed herein, cytoplasmic levels and nuclear levels of NMT1 canbe determined by a variety of means known in the art. For example, insome embodiments, microscopic analysis of at least one cell from thecell sample may be carried out. In these embodiments, the position ofNMT1 may be localized, for example, by antibody binding and subsequentimmunofluorescence and/or immunohistochemistry. In this manner, overalllevels of NMT1 as well as the cellular localization thereof can bedetermined. Furthermore, one of skill in the art can easily determine ifoverall levels of NMT1 are high in either the cytoplasm or nucleus of agiven cell for example by comparison with or simply based on knowledgeof a control. For example, such a control may be a “positive” controlfrom one or more cells known to have high cytoplasmic or nuclear levelsof NMT1 or a “negative” control from one or more cells known to have lowcytoplasmic or nuclear levels of NMT1.

As will be known by those of skill in the art, one method for carryingout such a determination is called an H or IHC Score. In methods such asthis, slides are scored using standard light microscopy. For example,IHC scores are derived from assessment of both average stainingintensity across the two tumor cores (scale 0 to 3) and percentage ofpositive cells (0 to 100%). These two scores, when multiplied, generatean IHC or H-score of 0 to 300. An “H” score higher than 100 isconsidered high and less than 100 is considered low for nuclear NMT1,whereas, an “H” score higher than 150 is considered high and lower than150 is considered low for cytoplasmic NMT1.

In some embodiments, if cytoplasmic levels of NMT1 are determined to behigh, for example, having an H score or IHC score of greater than 150,as discussed above, this indicates that the response of the patient toendocrine therapy will be poor, meaning that the patient is at risk forcancer recurrence and death due to breast cancer. Furthermore, a patientwith this prognosis would be given a systemic cancer treatment, such asfor example chemotherapy, and monitored more frequently for possiblerecurrence, that is, would be scheduled for more frequent doctor visitsand/or examinations than would a patient with a “good” or “favorable”prognosis as understood and accepted by those of skill in the art.

In some embodiments, if nuclear levels of NMT1 are determined to behigh, that is, for example, an H score of greater than 100, as discussedabove, this indicates that the endocrine therapy response of the patientis likely to be good, and that the patient is at significantly lowerrisk of recurrence and death due to breast cancer. Accordingly, apatient with this outcome can be administered endocrine therapy andmonitored less frequently for possible recurrence.

If both nuclear and cytoplasmic NMT1 levels are low (for example, an Hscore less than 100 or less than 150 respectively), the endocrinetherapy response is worse, with significantly higher risk or rate ofrecurrence and death due to breast cancer. A patient with this outcomeshould be assigned a more aggressive systemic therapy than endocrinetherapy and monitored much more frequently, as discussed above.

According to another aspect of the invention, there is provided a methodof determining the prognosis of a breast cancer patient comprising:

-   -   extracting a cell sample from a breast tumor of a patient;    -   determining the cellular localization of NMT2 in at least one        cell of the cell sample;    -   wherein if the at least one cell of the cell sample is positive        for nuclear NMT2, the prognosis is poor, and    -   if the at least one cell of the cell sample is negative for        nuclear NMT2, the prognosis is good.

For example, a poor prognosis means that the patient is at risk ofrecurrence or death due to breast cancer within the first ten years ofdiagnosis and should be given systemic treatment (chemotherapy) andmonitored for recurrence more frequently, as discussed herein.

For a patient with a good prognosis, wherein there is no nuclear NMT2,the patient may be put on endocrine therapy (in the case ofhormone-positive cancers) alone for the first ten years and then justmonitored for the next ten years.

According to another aspect of the invention, there is provided a methodof determining the prognosis of a triple-negative breast cancer patientcomprising:

-   -   extracting a cell sample from a breast tumor of a patient;    -   determining the cellular localization of NMT2 in at least one        cell of the cell sample;    -   wherein if the at least one cell of the cell sample shows        positive nuclear localization of NMT2, the prognosis is poor.

If the prognosis is poor, most of the breast cancer patients will diewithin the first ten years after diagnosis, usually within the first 4years of diagnosis. These patients should be placed on increasedsurveillance.

As will be appreciated by one of skill in the art, if there is nonuclear localization of NMT2, the prognosis is better or favorable, asdiscussed herein. Endocrine therapy will be the adjuvant therapy to allhormone receptor breast cancer irrespective of the NMT2 status. Positivenuclear NMT2 staining will be suggestive of the poor prognosis andrecurrence wherein additional combination therapy along with endocrinetherapy may benefit the patient.

As discussed herein, after ten years, the nuclear NMT2 doesn't mattermuch but high cytoplasmic NMT2 still relates to a bad outcome.Specifically, the survival outcome is poor for very high NMT2 in thecytoplasm. This remains true as an intermediate predictor for high NMT2in the cytoplasm is a poor predictor.

As will be appreciated by one of skill in the art, while in someembodiments, at least one cell of the cell sample is examined, it ispreferable that a statistically significant number of cells are analyzedand the results analyzed, for example, averaged. In some embodiments, atleast about one hundred cells from the cell sample are examined fordetermining NMT1 and/or NMT2 levels and/or cellular location orsubcellular location as discussed herein.

According to another aspect of the invention, there is provided a methodof slowing progression of a breast cancer tumor or improving outcome ofa breast cancer treatment comprising:

-   -   administering an effective amount of an NMT inhibitor to a        patient having a cancerous breast tumor, thereby preventing        phosphorylation of NMT2 and subsequent nuclear localization of        NMT2.

In some embodiments, the NMT inhibitor is a compound that inhibits bothNMT1 and NMT2 activity.

In some embodiments, the NMT inhibitor is an NMT2 inhibitor, that is,specific for inhibition of NMT2 activity.

In some embodiments, the NMT2 inhibitor is an NMT2 serinephosphorylation inhibitor.

In some embodiments, the NMT2 serine residue at which phosphorylation isbeing inhibited is S38 or S68.

In some embodiments of the invention, the serine phosphorylationinhibitor blocks access to S38 or S68 for the kinase, for example, bybinding to a region of NMT2 encompassing S38 or S68 as discussed hereinand/or by disrupting the NMT2 nuclear localization signal (NLS).

In some embodiments of the invention, the NMT2 serine phosphorylationinhibitor is an antibody or a small molecule, as discussed herein.

As will be appreciated by one of skill in the art, since theintervention is at the N-terminus, the enzymatic activity of NMT2 is notinhibited as the catalytic domain is in the C-terminus

According to another aspect of the invention, there is provided a methodof identifying a compound capable of inhibiting nuclear translocation ofcytoplasmic NMT2 comprising:

-   -   in an in vitro system, growing a plurality of test cells under        conditions suitable for nuclear translocation of cytoplasmic        NMT2 in the presence of a compound of interest wherein but for        the presence of the compound of interest, cytoplasmic NMT2 will        migrate into the nucleus of a respective one cell of the        plurality of cells; and    -   determining if cytoplasmic NMT2 has translocated into the        nucleus of at least one of the plurality of test cells in the        presence of the compound of interest,    -   wherein if less cytoplasmic NMT2 has translocated into the        nucleus of the at least one representative cell of the plurality        of cells than translocated into the nucleus of at least one        control cell grown under similar conditions except for the        presence of the compound of interest, the compound of interest        inhibits nuclear translocation of cytoplasmic NMT2.

As will be appreciated by one of skill in the art, any suitable cellscould be used in such a method and these cells could be grown under anysuitable growth conditions, for example, under standard cell culturegrowth conditions.

In some embodiments of the invention, nuclear translocation of NMT2 isdetermined by measuring proliferation of the test cells, as cells thathave NMT2 in the nucleus become highly proliferative. As is known bythose of skill in the art, there are a large number of methods formonitoring and/or measuring cell proliferation, that is, cell growth,known in the art which can be used within this aspect of the invention.

The invention will now be further explained and/or elucidated by way ofexamples; however, the invention is not necessarily limited to or by theexamples.

Results obtained from PhosphoNet and PhosphoSitePlus databases revealedthat the most likely site of phosphorylation of NMT2 by mTOR is serine38 followed by serine 68. NLStradmus query results that the poly lysinesequence, which runs from position 46 to 59 in the NMT2 primarystructure, is likely acting as an NLS. Such a NLS would allow NMT2protein to couple to importin, a shuttling protein which allows for thepassage of protein across the cell's nuclear membrane. A Multiplesequence alignment performed by Maga 7.0 software affirms that both ofthe S38 and S68 phosphorylation sites and the NLS are highly conservedin the NMT2 amino acid sequence across various species including humans.This high level of conservation signifies the importance of these sitesin the regulation and/or function of NMT2. The sequence alignment alsorevealed a highly conserved poly lysine sequence from residues 46 to 59which is flanked by the S38 and S68. This suggest that phosphorylationof these serine residues could lead to conformational change of the NLSof NMT2 which may function to determine the interactions of NMT2 withnuclear pore shuttling proteins such as importin. It is plausible thatphosphorylation of site 38 or 68, or of both sites simultaneously, maybe regulating whether the NLS of NMT2 is exposed on the surface orembedded within the protein. If the NLS is exposed this would make itpossible for importin, a nuclear pore shuttling protein, to interactwith NMT2 and escort it over the nuclear membrane and into the nucleus.

Example 1—Expression of NMT2-GFP in MCF7 Cells

All NMT2 plasmid constructs were successfully transfected into MCF7cells. Transfected cells for MCF7-GFP-NMT2, MCF7-GFP-NMT2-S38A,MCF7-GFP-NMT2-S38E, MCF7-GFP-NMT2-S68A, MCF7-GFP-NMT2-S68E andMCF7-GFP-NMT2-K49E lines displayed the expression of GFP underfluorescent microscopy. The results obtained from fluorescent microscopyanalysis of these cell cultures excluded the S68A, S68E and K49E celllines from further research. To further confirm the presence of NMT2 GFPfusion protein in the three remaining transfected cell lines, whole celllysates were collected from each line, along with the MCF7 line, andanalyzed by Western blot. The analysis displayed the expression ofpolypeptide band at the 60 kDa region of the membrane for all fourlines. The 60 kDa region corresponds with the molecular weight ofendogenous NMT2 protein. The analysis also displayed polypeptide bandsat the 87 KDa region of the membrane in all three NMT2 GFP transfectedcell lines. This region corresponds with the molecular weight ofNMT2-GFP and confirmed the presence of NMT2 GFP in the three transfectedlines.

Example 2—Sub Cellular Expression Patterns of NMT2 GFP

To determine if NMT2 phosphorylation status at serine 38 and 68 residuesregulates its subcellular localization, NMT2-GFP fusion protein serine38 or 68 residues were changed to either alanine residues (phospho-dead)or glutamic acid residues (phospho-mimic). A lysine residue at position49 in the NMT2 putative NLS was also changed to a glutamic acid residueto determine if disruption of the NLS would affect NMT2 localization.All forms of the NMT2 GFP fusion protein, including non-mutated NMT2GFP, were expressed in MCF7 ER+ breast cancer cells.

Disruption of the NMT2 NLS did appear to result in no nuclearlocalization of the NMT2 K49E GFP as expected.

As for the serine 38 mutations, when the residue was mutated to analanine, NMT2-GFP showed no nuclear localization. However, when theresidue was mutated to a glutamic acid, nuclear localization of theNMT2-GFP was greatly increased. Many S38E cells show very prominentexpression of the NMT2 GFP inside their nuclei. Results from fluorescentmicroscopy of MCF7-GFP-NMT2-S38A or MCF7-GFP-NMT2-S38E stained for—DAPIshow direct overlap of the expression of the NMT2 S38E GFP and thenuclei staining DAPI stain. Results from a Western blot analysis ofnuclear fractions for the MCF7-GFP-NMT2, S38A and S38E cell lines showstrong expression of NMT2-GFP in the nuclear fractions of the S38E lineand very little to no expression in the nuclear fractions of the S38Aline. These results together confirmed the presence of nuclear NMT2 GFPin the S38E line.

It appears that phosphorylation status at serine 38 regulates thesubcellular localization patterns of NMT2.

Example 3—Nuclear NMT2 Increases MCF7 Proliferation Rates

To determine if the constitutive phosphorylation of NMT2 protein playsany role in MCF7 cellular proliferation rates, two cellularproliferation assays were performed. In the trypan blue assay, by the 72hour time point, the S38E cell line had grown from 12,500 cells/ml ofmedia (50,000 cells total) to approximately 1.1 million cells/ml whilethe MCF7 and the MCF7-GFP-NMT2 control lines had only grown toapproximately 600,000 cells/ml and the S38A line had only managed togrow to approximately 400,000 cells/ml. The S38E line proliferated atnearly twice the rate of the control lines and more than twice the rateof the S38A line. A t-test statistical analysis found a significantdifference between the growth rates of the MCF7 and S38E and cell lineswith a P-value of 0.002 at the 72-hour time point (double asterisks inFIG. 5 ) and a P-value of 0.05 at the 96-hour time point (singleasterisk). This significance likely decreased (from 0.002 to 0.05) astime elapsed from 72 to 96 hours due to lack of space in the S38E wellscausing cells to die and approach MCF7 cell counts. A significantdifference was also found between the proliferation rates of the S38Aand MCF7 cell lines at the 96-hour time point (double asterisks) with aP-value of 0.02. Also, to be noted, the S38E cell line was the firstline to obtain enough cellular mass to reach the point of massivecellular death due to a lack of floor space in the culture plate and alack of nutrient in the media, confirming that the S38E line grew andproliferated the quickest.

Results for the crystal violet assay followed a similar tread. Again, atthe 72- and 96-hour time points, the S38E line was proliferatingsignificantly faster than the two control lines. The S38A line alsoappeared to be proliferating more slowly than the two control lines. At-test statistical analysis revealed a significant difference betweenthe proliferation rate of the MCF7 and S38E cell lines at the 72- and96-hour time points with P-values of 0.05 (single asterisk in FIG. 6 )and 0.003 (double asterisk in FIG. 6 ) respectively. The significanceincreased (from 0.05 to 0.003) as time elapsed from 72 to 96 hours dueto the faster growth rate of the S38E cell line. Based on the results ofthese proliferation assays, it appears that preventing NMT2phosphorylation at site 38 slows the proliferation rate of MCF7 cellswhile constitutive phosphorylation of this site increases the rate. Itis plausible that NMT2, which has accessed the nucleus of the cell, mayplay a role in transcription regulation and thus may be contributing tothe regulation of proliferation.

Example 4—NMT2 Phosphorylation Status and the IGF (PI3K/Akt/mTOR)Pathway

One of the key regulating proteins of the insulin like pathway is thetransmembrane IGF1R protein. Activation of IGF1R leads to activation ofall downstream proteins of the pathway. Therefore, increased expressionof IGF1R could lead to increased activity in this pathway which in turncould lead to increased cellular proliferation rates.

Increased expression and/or hyperactivation of IGF1R is one of thehallmark features of breast cancers and is being pursued as atherapeutic target. Therefore, RT-qPCR was performed to determine therelative expression rates of the IGF1R transcript in MCF7,MCF7-GFP-NMT2, MCF7-GFP-NMT2-S38A and MCF7-GFP-NMT2-S38E cell lines(FIG. 5 ). Relative to MCF7 cells, the results show increased expressionof IGF1R transcript in the S38E line and an under expression of thetranscript in the S38A line. The transcription rates of IGF1R geneappear to be greater in the MCF7 cells which are expressingconstitutively phosphorylated, at serine 38, NMT2 protein. The MCF7cells which are expressing NMT2 which cannot be phosphorylated at serine38 are showing lower transcription rates.

Example 5—NMT2 Localization Status and MCF7 Metastasis

MCF7 cells are normally considered to be weakly metastatic (R. B. Hazanet al., The Journal of Cell Biology, vol. 148, no. 4, pp. 779-790,February 2000).

Investigation into the expression patterns of tight junction relatedclaudin family proteins was initiated for the MCF7, MCF7-GFP-NMT2,MCF7-GFP-NMT2-S38A and MCF7-GFP-NMT2-S38E cell lines. Protein expressionlevels for five members of the claudin family, claudin 1-5, wereanalyzed by Western blot. No distinctive patterns of expression wereobserved for any of the claudins except for claudin 1. A previous studysuggested that decreased expression of claudin 1 protein may play a rolein breast cancer invasion and metastasis and that low claudin 1expression closely correlates with recurrence status in breast cancer(S. Morohashi et al., International Journal of Molecular Medicine, vol.20, no. 2, pp. 139-143, August 2007). Western blot analysis of claudin 1protein revealed an increase in expression of the protein in the S38Aline relative to MCF7 cells. A t-test statistical analysis of thewestern blot data showed a significant difference (P-value=0.018) inclaudin 1 protein expression between the S38A and S38E lines. Thisindicates that when NMT2 is blocked from entering into the nucleus,claudin 1 expression increases. Thus, it can be deduced that if NMT2 isallowed to enter into the nucleus, as is the case in the S38E line, thena decrease in claudin 1 expression can occur. The increased expressionof claudin 1 in the S38A line may help explain why clumped growthpatterns are observed. It is possible the blocking NMT2 from enteringinto the nucleus, by blocking phosphorylation of serine 38, may bepreventing NMT2 from playing its normal role in regulation of the CLDN1gene. This data revels a possible link between NMT2 regulation andcancer cell metastasis.

Example 6—Relevance of NMT1 in Clinical Outcome of ER Positive BreastCancer

ER positive breast cancer cells develop resistance to SERMs partly dueto activation of PI3K/Akt pathway. Recently, it has been demonstratedthat activated mTOR is associated with better clinical outcome inprimary tumors from an ERα positive cohort of breast cancer patients whowere subsequently treated with tamoxifen (Shrivastav A et al., BreastCancer Res 2014, 16(3):R49). Cytoplasmic and nuclear expression in abovebreast cancer cases was determined and an H score was given based on theintensity and percent positive cells for NMT1. Nuclear NMT1 predictedbetter recurrence free survival or recurrence free survival and deathdue to breast cancer.

FIG. 1 shows that high levels of nuclear NMT1 correspond to longerrelapse free survival while low levels of both cytoplasmic and nuclearNMT1 correlate to shorter relapse free survival. As depicted in Table 1,patients with both low cytoplasmic and nuclear levels had a hazard ratioof 1.49 whereas patients with high levels of only nuclear NMT1 had thelowest hazard ratio 0.70. Patients with high cytoplasmic had a hazardratio of 0.79 however this was not statistically significant. Togetherthese data suggest that NMT1 could serve as a prognostic marker forpredicting the possibility of recurrence.

In FIG. 2 , it is evident that high levels of nuclear NMT1 are linked tolower levels of death due to breast cancer or recurrence.

Upon analysis of Table 2, it is evident that high levels of nuclear NMT1had the lowest hazard ratio. As previously seen in the recurrence data,low levels of NMT1, be it cytoplasmic or nuclear, corresponded to highhazard ratios. The other data in the table was not statisticallysignificant.

Three serine residues were identified that had high predictive scoresfor phosphorylation by either mTOR or the AKT isoforms. The serine 38and 68 residues were predicted to be phosphorylated by mTOR and S258 bythe three AKT isoforms. Interestingly, mTOR was the top ranked kinase topotentially phosphorylate S38, which is positionally analogous to S47 ofNMT1. These serine residues are closest to the N-terminal end of the NLScommon to both isozymes. This suggests that phosphorylation of theserine most proximal to the N-terminal end of the NLS of NMTs may befunctionally conserved feature, despite the fact that the isoformsdisplay sequence dissimilarity in the N-terminal regions.

Example 7—NMT1 and NMT2 Contain a Nuclear Localization Sequence (NLS)

Observation of a poly-lysine region within the two NMT proteins wasnotable as this sequence is indicative of an NLS (Kosugi, S., et al., JBiol Chem, 2009. 284(1): p. 478-85). Indeed, the NLStradamus softwarepredicted that both NMT1 and NMT2 contain an NLS. The predicted NLSspans from K55 to K67 within NMT1 and from K46 to K58 within NMT2. Thepredicted NLSs constitute the same poly-lysine regions originallyobserved. This poly-lysine may be required for the binding of NMT1 toimportin a, a nuclear pore protein that facilitates protein transportinto the nucleus.

Example 8—NMT1—S47A/S47E-GFP

MCF7 cells expressing NMT1 with the S47A mutation expressed the GFPfusion protein exclusively in the cytoplasm. Interestingly,NMT1-S47A-GFP cells rapidly outgrew non-expressing cells under selectionpressure of the G418 eukaryotic anti-biotic, constituting approximately80% of the tissue culture surface area. Cells expressing cytoplasmicNMT1-S47E-GFP were remarkably lower in number compared to rapidlyproliferating S47A counterparts under the same selection pressures, onlyproliferating at a rate that occupied approximately 5-10% of the cellculture surface area, with the remaining area consisting ofnon-transfected cells. A sub-population of cells expressed NMT1-S47E-GFPas 1-3 discernable spots overlapping the nuclear region of the cells,however, these fluorescence signals were subject to rapidphotobleaching. Thus, it was difficult to quantify the overallpopulation relative to cells expressing cytoplasmic fusion protein, ornon-expressing cells.

Example 9—MCF7 Gene Expression of NMT and WT1 Regulated Genes FollowingInsulin Stimulation

Growth conditions rich in serum growth factors and MCF7 cells withenhanced PI3K/AKT/mTOR activity both displayed increased NMT1-BASP1interaction, with the latter exhibiting this interaction enhanced in thenucleus. NMT1 was also shown to bind to regions of DNA containing BASP1regulated growth genes. Indeed, NMT1 and NMT2 expression showed littlechange following insulin or rapamycin treatment, suggesting that thePI3K/mTOR/AKT signalling pathway primarily regulates NMT1 and NMT2through post-translational mechanisms. This supports our prediction thatphosphorylation is the leading PI3K/AKT/mTOR mediated regulatorymechanism responsible for NMT1 modulation.

Example 10—Nuclear Localization Sequence (NLS) Identification

The nuclear import of NMT2 is aided by a nuclear transporter protein,importin, which escorts the target cargo protein through a nuclear porecomplex into the nucleus.

Example 11—Differential Growth Rates of MCF7 and MCF7 NMT2 MutantExpressing Cells

In order to make ample stock of transfected MCF7 cells, they werecultured and expanded by incubating at 37° C. in 5% CO₂. However, duringthe cell culture it was noticed that MCF7-GFP-NMT2-S38A andMCF7-GFP-NMT2-S38E cells displayed very different proliferation ratescompared to MCF7 and MCF7-GFP-NMT2 cells.

On day 1, the cells from each of the four lines were easily containedwithin the floor space of one 100/20 mm culture dish. All four of thelines were approximately 30-40% confluent. On day three, the MCF7,MCF7-GFP-NMT2, and S38E lines were split into three fresh culture dishesas the cells were confluent. The MCF7 and MCF7-GFP-NMT2 lines wereapproximately 80% confluent at the time of passaging and the S38E linewas approximately 90% confluent. The S38A line was still atapproximately 65% confluency. By day seven, the S38E line was confluentand had to be passaged again into four dishes. The MCF7 andMCF7-GFP-NMT2 lines were at approximately 65% confluency and so were notyet confluent enough to be passaged. Surprisingly, the S38A line wasstill not confluent in its original culture dish. The S38E cellsappeared to be proliferating faster than the MCF7 and MCF7-GFP-NMT2lines and much faster than the S38A line which appeared to beproliferating slower than the MCF7 and MCF7-GFP-NMT2 lines. Theseseemingly differing growth patterns warranted further investigation inthe form of proliferation assays.

Example 12—Trypan Blue and Crystal Violet Proliferation Assays

Cellular proliferation rates were analyzed for MCF7, MCF7-GFP-NMT2,MCF7-GFP-NMT2-S38A and MCF7-GFP-NMT2-S38E cell lines using trypan blueand crystal violet proliferation assays. In the trypan blue assay (FIG.5 ), at time point zero, all wells for all four cell lines containedapproximately 50,000 cells.

At the 72-hour time point, the S38A cell line had proliferated toapproximately 400,000 cells/ml while the MCF7 and MCF7-GFP-NMT2 controllines were at approximately 600,000 cells/ml. The S38E cell line hadproliferated to approximately 1.1 million cells/ml. Between the 72- and96-hour time points, the S38E cells rapidly became over confluent andstarted to undergo cell death due to lack of space to grow further andthus show a plateau in the curve. Due to the rapid proliferation of S38Ecells, it became the rate limiting factor in carrying the assay post 96h. At the 96-hour time point the S38E line was at approximately 1.25million cells/ml while the S38A line was still only at approximately500,000 thousand cells/ml.

The crystal violet assay (FIG. 6 ) relies on the amount of cell biomass(i.e. DNA), rather than the number of cells, as the dye binds to ribosesugars. The assay was performed in a 24 well plate with an equal numberof cells (30,000) from each of four lines in each well. At time pointzero, absorbance readings ranged from 0.24-0.42. Due to a dilutionerror, the S38A wells may have contained more than the intended 30,000cells, and appeared to be approximately 20% more confluent in cellnumber (viewed under white light microscope) when compared to the wellsof the other three cell lines which all appeared to containapproximately the same cell confluency. The S38A line had the highestabsorbance rating of approximately 0.42 as a result of this dilutionerror. At the 96-hour time point, the S38A line had an absorbancereading of 1.63 nm while the S38E line had an absorbance rating of 3.74nm. The two control lines showed a very similar proliferation ratethroughout the assay. At the 96-hour time point the MCF7 line had anabsorbance reading of 1.7 nm and the MCF7-GFP-NMT2 line had a reading of2.3 nm.

Example 13—IGF1R Gene and Protein Expression Patterns

Dysregulation of the PI3K/Akt/mTOR pathway has been implicated in thedevelopment and progression of many types of cancer including ER+ breastcancer. The receptor tyrosine kinase transmembrane receptor responsiblefor the activation of this pathway is IGF1R. PhosphoNet software wasused to determine the feasibility of the phosphorylation of NMT2, atposition 38 and 68 serine residues, by mTOR, or any member, of thePI3K/Akt/mTOR pathway. Of the 500 human kinases analyzed by Phosphonet,mTOR received the highest kinase predictor V2 proximity score of 460,meaning that of the 500 kinases analyzed, mTOR is the most feasible forthe phosphorylation of NMT2 at position 38. For the serine at position68 mTOR did not receive a kinase predictor V2 proximity score.Interestingly, serine 68 was predicted to be phosphorylated by p70S6Kprotein, a downstream target of mTOR in the PI3K/Akt/mTOR pathway, witha kinase predictor proximity score of 1807.

RT-qPCR was performed to determine the status of IGF1R gene expressionin MCF7, MCF7-GFP-NMT2, MCF7-GFP-NMT2-S38A and MCF7-GFP-NMT2-S38E celllines (FIG. 23 ). The results show an under expression of IGF1R mRNA inS38A cells and an overexpression of IGF1R mRNA in S38E cells relative tothe MCF7 control line.

IGF1R protein expression levels for all four lines were determined byWestern blot analysis. The Western blot analysis of IGF1R is depicted inFIG. 8 (one of three Westerns which were performed for IGF1R proteinexpression analysis). All three independent experiments showed a similarpattern of expression for all four lines. The IGF1R protein expressionlevels obtained from these Western blots correlate closely with thepattern of expression shown in the previous IGF1R RT-qPCR chart. TheWestern blots show a very faint band of IGF1R protein expression in theS38A line while the RT-qPCR shows an under expression of IGF1Rtranscript in this line. A much stronger band of expression appears inthe S38E line on the Western blot and the RT-qPCR displayed anoverexpression of IGF1R transcript in this line.

The S38A cell line expressed a very low level of normalized IGF1R totalprotein while the S38E line expressed a high level IGF1R total protein.

Example 14—Localization of NMT2 in (A) MCF7 Cells and (B) MDA-MB 231

With reference to FIGS. 9A and 9B, it can be seen that the expression ofNMT2 was mostly observed in the cytoplasm of MCF7 cells. The MCF7 cellline was established from a pleural effusion at the Michigan CancerFoundation. As the cells were originally derived from the metastases ofan advanced tumor, the cell line is non-invasive. The MCF7 cellsrepresents an early-stage disease because of the presence of functionalER and its dependence on estrogen for growth both in vitro and in vivo(JoEllen Welsh, Chapter 40—Animal Models for Studying Prevention andTreatment of Breast Cancer, Editor(s): P. Michael Conn, Animal Modelsfor the Study of Human Disease, Academic Press, 2013, Pages 997-1018).In contrast, NMT2 was mostly localized in the nucleus of MDA-MB-231cells. The MDA-MB-231 cell line was established from a pleural effusionof a patient with invasive ductal carcinoma. The MDA-MB-231 cellsrepresents a late-stage disease. This cell line expresses mutated p53and is ER, PR, and E-cadherin negative. These observations furthervalidate the premise that nuclear NMT2 is potentially a hallmark featureof metastatic and invasive cancers.

Example 15—the Subcellular Localization of NMT1 in Hormone Positiveand/or Her2Nue Positive Breast Cancers

With reference to Tables 3-5 and FIGS. 10-13 , it can be seen that thelocalization of NMT1 in the cytoplasm and/or nucleus in the primarybreast cancer tissue provides the prognosis or the treatment response toendocrine therapy. The high expression of NMT1, semi-quantitativelydetermined by the “H” score, in the nucleus corresponds to a betterprognosis, that is, with predicted better treatment outcomes when thesepatients later underwent endocrine therapy, whereas high cytoplasmicexpression of NMT1 predicted poor prognosis and poor treatment response.Furthermore, both low cytoplasmic and low nuclear expression predictedworse prognosis and treatment response. As discussed herein, the statusof the NMT1 expression will aid oncologists to identify the patientsthat were predicted to have poor prognosis and treatment response to dueto the status of NMT1 in their breast cancer tissues. With thisknowledge, the oncologists may put patients predicted to have poorprognosis and endocrine therapy response on regular surveillance anddesign treatment regimens that may include combination therapy orsystemic chemotherapy to manage breast cancer and avoid recurrence.

Similarly, the localization of NMT2 in the nucleus is an indicator ofhighly invasive and metastatic breast cancer. The breast cancer tissuesshowing positive nuclear staining leads to poor survival. The nuclearlocalization of NMT2 was observed to be positive in cases of death dueto breast cancer and recurrence. Most of the nuclear positive cases wereobserved in the triple negative breast cancer cohort, while survivalfrom all the breast cancer types suggested poor outcome and recurrencein cases where the NMT2 was observed to be localised in the nucleus.High expression of cytoplasmic NMT2 also indicated poor prognosis andtreatment outcomes with greater mortality even in those patients whosurvived for more than 10 years. As discussed herein, knowledge of thestatus of nuclear NMT2 in the primary breast cancer tissues would allowoncologists to design breast cancer treatment regimens wherein thenuclear NMT2 staining is positive. Specifically, breast cancer patientswith nuclear NMT2 will be monitored more frequently and may beprescribed combination therapy that may include systemic chemotherapy.The status of NMT1 and NMT2 may be useful in deciding which hormonepositive breast cancer patient should remain on endocrine therapy beyondfive years rather than putting everyone on endocrine therapy for 10years or more.

While the preferred embodiments of the invention have been describedabove, it will be recognized and understood that various modificationsmay be made therein, and the appended claims are intended to cover allsuch modifications which may fall within the spirit and scope of theinvention.

TABLE 1 Univariate analysis of factors associated with recurrence freesurvival Hazard Single Predictor Number Ratio 95% CI on HR P NMT1Nucl >100 440 0.70 0.510 to 0.97 0.0304 NMT1 Cycto >150 440 0.79 0.580to 1.08 0.1347 Both Nuclear & 440 1.49 1.08 to 2.04 0.0014 CytoplasmicLow

TABLE 2 Univariate analysis of factors associated with death due tobreast cancer or recurrence Hazard Single Predictor Number Ratio 95% CIon HR P NMT1 Nucl >100 440 0.71 0.530 to 0.97 0.0306 NMT1 Cycto >150 4400.75 0.550 to 1.01 0.0543 Both Nuclear & 440 1.54 1.540 to 2.08 0.0055Cytoplasmic Low

TABLE 3 First 120 months Event Death Hazard 95% Predictors N Event PRatio Limits Confidence Cyto 16-21 437 160 High >360 0.0284 1.72 1.062.81 Med 360 0.6854 0.93 0.65 1.33 Low <360 reference Age at Dx 0.00091.03 1.01 1.06 Nuclear 16-21 437 160 High >0 0.0006 1.74 1.27 2.39 LowZero reference Age at Dx 0.0002 1.04 1.02 1.06 Event Death or RecurrHazard 95% Predictors N Event P Ratio Limits Confidence Cyto 16-21 437192 High >360 0.0657 1.52 0.97 2.38 Med 360 0.3918 0.87 0.63 1.20 Low<360 reference Age at Dx 0.0007 1.03 1.01 1.05 Nuclear 16-21 437 192High >0 0.0027 1.56 1.17 2.09 Low Zero reference Age at Dx 0.0002 1.031.02 1.05

TABLE 4 from 120 months onwards. Event Death Hazard 95% Confi-Predictors N Event P Ratio Limits dence Cyto 172 65 High >360 0.89 1.060.48 2.35 Med 360 0.009 2.15 1.21 3.82 Low <360 reference Age at Dx<.0001 1.07 1.05 1.10 Nuclear 16-21 172 65 High >0 0.19 0.70 0.41 1.19Low Zero reference Age at Dx <.0001 1.07 1.04 1.09 Cyto 147 59 High >3600.68 0.82 0.33 2.07 Med 360 0.0031 2.46 1.35 4.46 Low <360 reference Ageat Dx <.0001 1.08 1.05 1.12 Nuclear 147 59 High >0 0.0366 0.55 0.31 0.96Low Zero reference Age at Dx <.0001 1.07 1.04 1.11

TABLE 5 Survival after Recurrence All subjects who were used had arecurrence. Time = 0 at recurrence so the whole spectrum of data isused. Cyto 148 123 High >360 0.031 1.85 1.06 3.25 Med 360 0.71 1.08 0.711.65 Low <360 reference Age at Dx 0.66 0.99 0.97 1.02 Nuclear 148 123High >0 0.0008 1.90 1.31 2.77 Low Zero reference Age at Dx 0.82 1.000.98 1.02

The invention claimed is:
 1. A method of determining the prognosis of abreast cancer patient comprising: extracting a cell sample from a breasttumor of a patient; determining the cellular localization of NMT2 in atleast one cell of the cell sample; wherein if the at least one cell ofthe cell sample is positive for nuclear NMT2, the prognosis is poor, andif the at least one cell of the cell sample is negative for nuclearNMT2, the prognosis is good.
 2. The method according to claim 1 whereina statistically significant number of cells are analyzed and the resultsare averaged.
 3. The method according to claim 1 wherein, in a poorprognosis, a prognosis score for the hormone breast cancer patient isdecreased.
 4. The method according to claim 1 wherein in a goodprognosis, a prognosis score for the hormone breast cancer patient isincreased.
 5. A method of determining the prognosis of a triple-negativebreast cancer patient comprising: extracting a cell sample from a breasttumor of a patient; determining the cellular localization of NMT2 in atleast one cell of the cell sample; wherein if the at least one cell ofthe cell sample shows positive nuclear localization of NMT2, theprognosis is poor.
 6. The method according to claim 5 wherein astatistically significant number of cells are analyzed and the resultsare averaged.
 7. The method according to claim 5 wherein, in a poorprognosis, a prognosis score for the hormone breast cancer patient isdecreased.
 8. The method according to claim 5 wherein in a goodprognosis, a prognosis score for the hormone breast cancer patient isincreased.